USP5-Beclin 1 axis overrides p53-dependent senescence and drives Kras-induced tumorigenicity

Non-small cell lung cancers (NSCLC) frequently contain KRAS mutation but retain wild-type TP53. Abundant senescent cells are observed in premalignant but not in malignant tumors derived from the Kras-driven mouse model, suggesting that KRAS oncogenic signaling would have to overcome the intrinsic senescence burden for cancer progression. Here, we show that the nuclear Beclin 1-mediated inhibition of p53-dependent senescence drives Kras-mediated tumorigenesis. KRAS activates USP5 to stabilize nuclear Beclin 1, leading to MDM2-mediated p53 protein instability. KrasG12D mice lacking Beclin 1 display retarded lung tumor growth. Knockdown of USP5 or knockout of Becn1 leads to increased senescence and reduced autophagy. Mechanistically, KRAS elevates ROS to induce USP5 homodimer formation by forming the C195 disulfide bond, resulting in stabilization and activation of USP5. Together, these results demonstrate that activation of the USP5-Beclin 1 axis is pivotal in overriding intrinsic p53-dependent senescence in Kras-driven lung cancer development.

WT). The results of manipulating USP-Beclin1 axis in both cell lines are the same. Does this mean that the USP-Beclin1 axis functions in lung cancer cell regardless of KRAS mutation? If so, please explain how.
3. Similarly, it would be important to define the role of the axis in p53 WT and p53-mutant/ko tumor cells, regardless of KRAS mutation.
4. The authors show nicely in vivo ubiquitination assay results in Fig3. Please explain whether this assay was performed under denaturing conditions. From the Methods in the manuscript, it seems the assay was performed with a normal Co-IP protocol, and not under denaturing conditions. As the results from ubiquitination assay are key parts of mechanism work, it will be important to explain and motivate in detail that how the in vivo ubiquitination assay was performed.
5. Then, an in vitro ubiquitination assay related to the Fig 3 should be performed.
6. The Co-IP bands in Fig4m are distinctly and clearly displayed. The bands for p53 and IgG should be very similar in size. Thus, how did you technically go about separating them? 7. In Fig5a and c, overexpression of KRAS-G12V upregulated the expression of USP5. By comparing the ratio between HA-Ub-Vs-USP5 and USP5, it seems that the increased USP5 activity was caused by increased protein expression of USP5. Hence, it seems a stretch to conclude that "KRAS-G12V significantly increased USP5 deubiquitinating enzymatic activity". In Fig5b, ROS inducers, H2O2, and PL clearly increased USP5 activity by comparing the ratio between HA-Ub-Vs-USP5 and USP5. The addition of NAC can't change the USP5 ratio. The authors should explain this further. It would also be informative if the authors include an experiment where cells that do not overexpress KRAS-G12V are incubated with NAC.
8. In Fig5I, shortly exposed bands in USP5-HA+KRAS-G12V sample should be added. If possible, significance levels should be analyzed in quantifications of CHX treatment results in Fig1b and c,Fig3a,Fig5d and I,and FigS4d. 9. The authors propose that USP5 and Beclin1 can regulate p53 stability and promote MDM2mediated p53 degradation. A ubiquitination assay for p53 should be performed to directly confirm MDM2-mediated proteasomal-dependent degradation of p53, for example, by depleting USP5 or Beclin1.
10. It is known that inhibition of USP5 induces apoptosis. Considering the role of p53 in cell apoptosis, how do authors exclude the possibility that the suppression of tumor growth by depletion of USP5 was not caused by increased apoptosis?
Reviewer #3 (Remarks to the Author): The manuscript by Juan Li et al discovered that USP5-Beclin 1 axis is critically involved in lung cancer cell senescence and tumorigenesis. Using a series of elegant experiments including cell biology, mechanistic studies, animal models and patient samples, the authors reported that USP5 stabilizes Beclin 1, which regulates p53 stability and senescence. Further, they showed that oncogenic Ras modulates USP5 via ROS, and that USP5-Beclin 1 axis is involved in lung tumorigenesis.
The main novelty of this study, in my opinion, is on USP5, and how USP5 regulates Beclin 1 in the context of lung cancer and oncogenic Ras mutations. The Beclin 1-p53 connections in senescence and cancer have already been reported.
A major strength of this manuscript is the rigor of the presented results. The observations are robust and supported by multiple models. With few exceptions, the experiments were designed in a meticulous manner, incorporating proper controls and statistical analyses throughout the study. This reviewer has the following suggestions: 1. The ubiquitination experiments presented in Fig. 2 g, h, i should be performed under denature IP condition. This is because native IP can bring down ubiquitin signals from interacting proteins and may not represent the ubiquitin from the IP'ed target.
2. While the authors showed co-IP results suggesting a Beclin 1-p53-MDM2 complex, the direct binding partner for Beclin 1 is unclear to this reviewer. These experiments can be strengthened by using in vitro translated proteins or purified protein fragments expressed from bacteria. I understand that these interactions could be facilitated by modifications in the cells. Either way, the authors are encouraged to clarify this.
3. Junying Yuan group reported that Beclin 1 deficiency leads to reduced p53 protein level (PMID: 21962518). The present study showed that disrupting Beclin 1 stabilizes p53 in lung cancer. How to reconcile these contradictory results? Minor: 1. There are several studies on USP5 in lung cancer (PMID: 34741014, PMID: 34858787, PMID: 32477134 and PMID: 30555744). The authors may consider mentioning some of these studies.

Typo in line 122 and line 149.
This review was submitted by Zhixun Dou.

Reviewer #1 Remarks to the Author:
This is an excellent an thoughtful manuscript that characterizes the relationship between autophagy and senescence in lung cancer development, The work further identifies interaction(s) between USP5, Beclin and p53 in the regulation of autophagy and senescence and raises the possibility that USP5 influences clinical outcomes in lung cancer patients. The studies presented utilize appropriate technical approaches, particularly cells with expression of recombinant/mutant proteins and shRNA silencing to interrogate the putative relationships and impact on senescence and clonogenic survival. These latter data are particularly robust and convincing of the tumor growth repressive impact of senescence.

Response:
We are grateful to the reviewer for the helpful comments and suggestions. We have addressed all of the issues raised by the reviewer.

Reviewer 1 Specific Comments #1:
A few deficiencies that likely could be readily corrected are indicated below.
"The Western blots should be accompanied by quantification of the protein bands. This is a problem, particularly in the case of LC3I to II conversion, where this indication of autophagy does not always appear to reflect the modulation of autophagy that the authors propose."

Response:
We sincerely appreciate the reviewer's suggestion. As suggested, we performed densitometry of the LC3-II protein bands and then normalized them to corresponding GAPDH protein bands as it was often used in the field (Nature, 2017, PMID: 28445460;Nat Med, 2016, PMID: 27841876). The graphs showed quantitative data derived from three independent western blot experiments (revised Figure 1b, 1c, 1f, 4j, 6a, 6d and revised Supplementary Figure S2d, S2g, S5j). In addition, we also examined the p62/SQSTM1 by western blot analyses or IHC (revised Figure 1b, 1c, 1f, 4j, 6a, 6d and revised Supplementary Figure S2d, S2g, S5j) and LC3 puncta by IF to reflect the modulation of autophagy (revised Figure 1a). The conclusions from the quantifications support our original conclusions.
Reviewer 1 Specific Comments #2: "A critical finding is that KRAS/G12D up regulates USP5 to promote autophagy, which prevents the cells from entering into senesence, and facilitates transformation. However, it appears that only a single time point is presented in support of this conclusion in Figure 5. This fundamental observation requires substantial confirmation."

Response:
To address this issue, we established stable H292 cells harboring doxycycline-inducible KRas G12V . We observed that induction of KRas G12V expression was readily detected after 48 hours upon doxycycline, concomitant with increased expression of USP5, Beclin 1, and LC3-II, as well as down-regulation of p53 and p62/SQSTM1 (revised Figure 5a), in keeping with our previous observations. Notably, the increased enzymatic activities of USP5 were clearly observed at 12 hours after KRas induction, concomitant with increased cellular ROS levels (revised Supplementary Figure S5a). Thus, the induction of KRas G12V expression leads to the elevation of ROS, and an increase of USP5 deubiquitinase activity, resulting in the modulation of downstream protein expression including Beclin 1, p53, p62/SQSTM1, and LC3-II, suggesting that KRas modulates USP5 deubiquitinase activity (revised manuscript, lines 290-296).
Reviewer 1 Specific Comments #3: "The authors refer to decreased p62 on line 221, but there is no reference to where this information could be found in a figure. It is further unclear why p62 levels are not assessed routinely, for instance in Figure 4j(find p62 WB). It should be noted that p62 is routinely referred to as SQSTM1."

Response:
We apologize for the unclear description and for the missed examination of p62 protein levels in the relevant experiments. The original statement "decreased p62 on line 221" could be found in the original Supplementary Figure S3b (revised Supplementary Figure S3c). Also, per suggestion, we used p62/SQSTM1 in the revised manuscript.
Regarding to the missing p62 protein levels, we performed a new set of western blotting using the same total cell lysates stored in -80 ℃ freezer. As shown in the revised Figures (Figure 1b, 1c, 1f, 6a, 6d and revised Supplementary Figure S2d, S2g, S5j), silencing of USP5 led to significantly increased p62/SQSTM1 protein expression, which could be effectively rescued by ectopic expression of Beclin 1, but not Beclin 1 S90+93A or Beclin 1 L184+187A , both of which are defective in promoting autophagy, indicating that silencing of USP5 inhibits autophagy in a cytoplasmic Beclin 1-dependent manner.

Response:
Our results indicate that activation of the USP5-Beclin1 axis can function to override p53-dependent senescence in lung cancer cells regardless of the KRAS status. In this study, we showed that an activating mutation of KRAS elevates intracellular ROS, leading to ROS-dependent oxidization and dimerization of USP5 protein through C195 disulfide formation, resulting in stabilization and activation of USP5 (revised Figure 5). Therefore, USP5 is a critical downstream effector of activated KRAS. Although we have not explored other means of activation of USP5, it is plausible that activation of USP5 via ROS can lead to modulation of Beclin 1-p53 axis in the absence of KRAS mutation (revised manuscript lines 300-310).

Reviewer 2 comment #4: "The authors show nicely in vivo ubiquitination assay results in Fig3
. Please explain whether this assay was performed under denaturing conditions. From the Methods in the manuscript, it seems the assay was performed with a normal Co-IP protocol, and not under denaturing conditions. As the results from ubiquitination assay are key parts of mechanism work, it will be important to explain and motivate in detail that how the in vivo ubiquitination assay was performed."

Response:
We apologize for the unclear description. The in vivo ubiquitination assays performed in the original Fig3 were not under denaturing conditions. As the reviewer suggested, we re-performed all of in vivo ubiquitination assays under denaturing conditions and we reached the same conclusions (revised Figure 2g, 2h, 2i and Supplementary Figure S4i, S4j, S5i).

Reviewer 2 comment #5: "Then, an in vitro ubiquitination assay related to the Fig 3 should be performed."
Response: We have repeated the in vitro ubiquitination assay under denaturing conditions (revised Figure 5l). showed that the deubiquitinating enzymatic activity of USP5 was elevated 12 hours after KRas induction, concomitant with increased cellular ROS levels, while the protein expression of Ras, USP5 and Beclin 1 were evidently elevated at 2 days after induction, together with reduced expression of p53 and p62/SQSTM1 in a time-dependent manner (revised Figure 5a). These results indicate that activation of KRas G12V leads to the elevation of ROS, resulting in increased deubiquitinating enzymatic activity of USP5 followed by the accumulation of USP5 protein (revised manuscript lines 290-297).
As the reviewer suggested, we treated H292 cells (KRAS WT ) with NAC. The results showed that treatment of NAC significantly decreased both basal levels of USP5 protein and activity (revised Figure S5c; revised manuscript lines 307-310).
Reviewer 2 comment #8: "In Fig5I, shortly exposed bands in USP5-HA+KRAS-G12V sample should be added. If possible, significance levels should be analyzed in quantifications of CHX treatment results in Fig1b and c,Fig3a,Fig5d and I,and FigS4d." Response: We appreciate the reviewer's suggestion. Accordingly, we added the shortly exposed bands (revised Figure 5m). Significance levels were analyzed in quantifications of CHX treatment results (revised Figure 2b, 2c, 3a, 5d, 5m, and Supplementary Figure S4e).
Reviewer 2 comment #9: "The authors propose that USP5 and Beclin1 can regulate p53 stability and promote MDM2-mediated p53 degradation. A ubiquitination assay for p53 should be performed to directly confirm MDM2-mediated proteasomal-dependent degradation of p53, for example, by depleting USP5 or Beclin1."

Response:
We sincerely appreciate the reviewer's suggestion. Accordingly, we examined the ubiquitination levels of p53 in either USP5-or Beclin 1-ablated 293FT cells transiently expressed p53-Myc and MDM2-HA. Ablation of USP5 or BECN1 significantly decreased the ubiquitin levels of p53, which could be completely rescued by ectopic expression of MDM2 (revised Supplementary Figures S4i and S4j).

Reviewer 2 comment #10:
"It is known that inhibition of USP5 induces apoptosis. Considering the role of p53 in cell apoptosis, how do authors exclude the possibility that the suppression of tumor growth by depletion of USP5 was not caused by increased apoptosis?" Response: To address this issue, we examined apoptosis in USP5-depleted A549 or H292 cells by western blotting and FACS analyses. Our results showed that the depletion of USP5 had little effect on the expression of apoptotic biomarkers (cleaved PARP or Caspase-3) (revised Supplementary Figure S2b). In addition, FACS analyses showed that depletion of USP5 barely caused apoptosis in A549 and H292 cells (revised Supplementary Figure S2c; revised manuscript lines 276-278).

Reviewer #3
Remarks to the Author:  Figure 4n-4o). Furthermore, Beclin 1 could facilitate the interaction between p53 and MDM2 in a dose-dependent manner in vitro (revised Figure 4q).

Reviewer 3 Comments #3:
"Junying Yuan group reported that Beclin 1 deficiency leads to reduced p53 protein level (PMID: 21962518). The present study showed that disrupting Beclin 1 stabilizes p53 in lung cancer. How to reconcile these contradictory results?" Response: We thank this reviewer for asking this question. We think that the different mechanisms may be due to the complex role of Beclin 1 in cancer development in a cancer-and cell-type-specific manner. Our study focuses on the cell types that can be induced to undergo senescence while most cancer lines cannot be induced to undergo senescence (therefore, they are already defective in senescence mediated by tumor suppression mechanism). Thus, the mechanisms revealed by this study may be relevant only to the cell types that can undergo senescence.
The notion that Beclin 1 can function as a tumor suppressor protein is supported by several well-known studies, including the observations that endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia (Nature,1999, PMID: 10604474); BECN1 is monoallelically lost in 40% to 75% of human breast, and ovarian cancers (Nature,1999, PMID: 10604474). The Becn1 +/mice are prone to the development of liver and lung tumors and lymphomas (J Clin Invest, 2003;PMID: 14638851;PNAS, 2003;PMID: 14657337).
However, large-scale genomic analyses of human cancers have failed to identify recurrent mutations in BECN1 (Nature, 2014, PMID: 24390350;Science, 2013, PMID: 23539594), implying that BECN1 may not be a classical tumor suppressor in most human cancers (Mol Cancer Res, 2014, PMID: 24478461). Additionally, BECN1 allelic loss is confounded by its location adjacent to BRCA1 on human chromosome 17q21, raising the possibility that loss of BECN1 in human cancers may be associated with the loss of BRCA1 in human breast and ovarian cancers (Mol Cancer Res, 2014, PMID: 24478461). Notably, in PALB2 loss mouse models for hereditary breast. cancer, allelic loss of Becn1 promotes p53 activation and reduces tumorigenesis (Cancer Discov, 2013, PMID: 23650262), suggesting an intimate relation of p53 in Beclin 1-mediated regulation of tumorigenesis.
Taken together, our study revealed the USP5-Beclin 1 axis in regulating p53-dependent senescence, which has not been addressed by previous studies (revised manuscript 446-452).

Reviewer 3 minor comment #1
"There are several studies on USP5 in lung cancer (PMID: 34741014, PMID: 34858787, PMID: 32477134 and PMID: 30555744). The authors may consider mentioning some of these studies."